Sirtinol was purchased from Sigma-Aldrich (St. Louis, MO, USA). Culture medium and its supplements including antibiotics and fetal bovine serum (FBS) were purchased from Gibco Invitrogen Corp. (Carlsbad, CA, USA). Primary antibodies against Atg5, Atg7, β-actin, beclin-1, cleaved caspase 7, caspase 7, cleaved caspase 9, Cdc2, cyclin A, cyclin B1, cyclin D1, cyclin E, cytochrome c, HDACs, LC3B and p21, and horseradish peroxidase-conjugated secondary antibody were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). Bax, Bcl-2, Cdk4, Cdk6, Cdk2, histone H1, poly(ADP-ribose) polymerase (PARP), p53, Ac-p53, and p27 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA); The Annexin V-FITC apoptosis detection kit I was from BD Biosciences (San Diego, CA, USA). All other chemicals were purchased from Sigma-Aldrich. Sirtinol was dissolved in dimethyl sulfoxide (DMSO) and stored at −20°C until use. Sirtinol was diluted to the appropriate concentrations in culture medium containing 1% FBS. The final concentration of DMSO was <0.1% (vol/vol), and was also present in the corresponding control.

MCF-7 human breast cancer cells were purchased from the American Type Culture Collection (Manassas, VA, USA). The cells were grown in Dulbecco’s modified Eagle’s medium (Gibco, Rockville, MD, USA) containing 10% heat-inactivated FBS. Cells were maintained as monolayers in a humidified atmosphere containing 5% CO₂ at 37°C and the culture medium was replaced every two days. After 48 h of incubation, the culture medium was replaced with treatment medium containing the desired concentrations of chemicals.

Cell viability was determined using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, 5 mg/ml, Sigma). The cultures were initiated in 96-well plates at a density of 2.5×10³ cells per well. After 48 h of incubation, the cells were treated with various concentrations of sirtinol and cultured for 24 or 48 h. At the end of the treatment period, 100 μl of 1X MTT reagent were added to each well and incubated for 4 h at 37°C in the dark. After incubation, the supernatant was aspirated and formazan crystals were dissolved in 100 μl of DMSO at 37°C for 15 min with gentle agitation. The absorbance per well was measured at 540 nm using the VersaMax Microplate reader (Molecular Devices Corp., CA, USA). Data from three independent experiments were analyzed and then normalized to the absorbance of wells containing medium only (0%) and untreated cells (100%). IC₅₀ values were calculated from sigmoidal dose-response curves using SigmaPlot 10.0 software.

MCF-7 cells were treated with sirtinol (2, 10, or 50 μM) for 48 h. Cells were harvested by trypsinization and washed twice with cold phosphate-buffered saline (PBS). For total protein isolation, cells were suspended in PRO-PREP™ protein extract solution (Intron, Seongnam, Korea) and protein concentrations were measured using the protein assay kit (Bio-Rad, Hercules, CA, USA) according to the manufacturer’s instructions. Equivalent amounts of proteins were resolved and electrophoresed using SDS-PAGE on a 6–15% gel. Gels were transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, Billerica, MA, USA) and membranes were blocked with blocking buffer (TNA buffer containing 5% skim milk) for 1 h. Subsequently, the membranes were incubated with primary antibodies at 4°C overnight. After washing for 1 h with TNA buffer (10 mM Tris-Cl, pH 7.6, 100 mM NaCl and 0.5% Tween-20), the membranes were incubated with horseradish peroxidaseconjugated anti-mouse or anti-rabbit antibody (1:10,000, Santa Cruz Biotechnology) for 30 min at room temperature and then washed for 1 h with TNA buffer. The blots were developed using an enhanced chemiluminescence (ECL)-plus kit (Amersham Biosciences, Buckinghamshire, UK).

The cells were treated with various concentrations of sirtinol (2, 10, or 50 μM) for 48 h. The total number of cells, including the ones in suspension and those adhered on the walls, were harvested separately for sub-G1 or other cell cycle stages, respectively, and washed in 1% bovine serum albumin (BSA) before fixing in 95% ice-cold ethanol containing 0.5% Tween-20 for 1 h at −20°C. The cells (1×10⁶) were washed in 1% BSA, stained with cold propidium iodide (PI) staining solution (10 μg/ml PI and 100 μg/ml RNase in PBS) and incubated in the dark for 30 min at room temperature. Data acquisition and analysis were carried out using a flow cytometry system (Becton-Dickinson, San Jose, CA, USA).

Morphological changes in the nuclear chromatin of apoptotic cells were identified by staining with the DNA binding dye DAPI. Cells were grown in 6-well plates at a density of 1×10⁵ cells per well followed by the desired treatment. After 48 h of incubation, the cells were washed with cold PBS, fixed with methanol for 30 min, rewashed and stained with 200 μl of DAPI solution (1 μg/ml) at 37°C for 30 min. After the staining solution was removed, the apoptotic cells were visualized using a fluorescence confocal microscope at ×400 magnification.

The Annexin V-FITC binding assay was performed according to manufacturer’s instructions using the Annexin V-FITC detection kit I (BD Biosciences). The cells were treated with sirtinol (2, 10, or 50 μM) for 48 h. The total number of cells was counted by trypsinization and washed twice with cold PBS. The cell pellet was resuspended with 100 μl of binding buffer at a density of 1×10³ cells per ml and incubated with 5 μl of FITC-conjugated Annexin V and 5 μl of PI for 15 min at room temperature in the dark. A total of 400 μl of 1X binding buffer was added to each sample tube and the samples were immediately analyzed by FACSCalibur (Becton-Dickinson).

The cultures were initiated in 6-well plates at a density of 1×10⁵ cells per well. Cells were allowed to attach for 48 h and exposed to SAHA for 48 h. Caspase 8 and caspase 9 activities in the cell lysate were measured using colorimetric assay kits (Biovision Ins., CA, USA) as described in the manufacturer’s protocol. The kits used in this study utilized synthetic tetrapeptides labeled with p-nitroanilide (pNA). Briefly, fifty microliters (100 μg) of cell lysate were incubated with 50 μl of 2× reaction buffer and 2 μl of IETD-pNA for capase 8 or LEHD-pNA for caspase 9 at 37°C for 2 h. A reading was then taken from a spectrophotometer at 405 nm with a VERS Amax Microplate Reader (Molecular Devices Corp.). In case of caspase 8 activity, a reading was taken from a fluorescence microtiter plate reader. Caspase 7 activities in the cell lysate were measured using caspase 7 immunoassay kits (Biovision Ins., CA, USA) as described in the manufacturer’s protocol. Briefly, the assay utilizes caspase 7 polyclonal antibody to capture activated caspase 7 from cell lysates. Substrate DEVD-AFC is then added that is cleaved proportionally to the amount of activated caspase 7 in the cell lysate. The cleavage generates free AFC which is then analyzed fluorometrically (Ex./Em. = 400/505 nm) using a fluorescence plate reader. The assay ensures absolute specific detection of caspase 7. Other known caspases and non-specific proteases are not detected.

MCF-7 cells were seeded in T-25 flasks and at 70% confluence the cells were treated with sirtinol for 48 h. At the appropriate time-points, cells were incubated with acridine orange (1 μg/ml) in serum-free medium at 37°C for 15 min. The acridine orange was removed and fluorescent micrographs were obtained using an inverted fluorescence microscope (Olympus FV10i; Olympus, Tokyo, Japan). The cytoplasm and nucleus of the stained cells fluoresced bright green, whereas the acidic autophagic vacuoles fluoresced bright red. Cells were treated with 200 nmol/l bafilomycin A1 for 30 min before the addition of acridine orange to inhibit the acidification of autophagic vacuoles. To quantify the development of acidic vesicular organelles (AVOs), sirtinol-treated or control cells were stained with acridine orange (1 μg/ml) for 15 min and removed from the plate with trypsin-EDTA, and collected in phenol red-free growth medium. Green (510–530 nm) and red (650 nm) fluorescence emission from 1×10⁴ cells illuminated with blue (488 nm) excitation light were measured with a FACSCalibur.

Autophagic vacuoles were also detected with MDC by incubating the cells with MDC (50 μmol/l) in PBS at 37°C for 10 min. After incubation, the cells were washed four times with cold PBS and fixed with 3.75% paraformaldehyde in PBS. The cells were immediately analyzed by fluorescence microscopy using an inverted microscope (Olympus FV10i) equipped with a filter system (excitation wavelength, 380 nm; emission filter, 525 nm).

We first assessed the cytotoxicity of sirtinol in MCF-7 cells by using the MTT assay. The chemical structure of sirtinol is shown in Fig. 1A. Sirtinol significantly reduced the growth of MCF-7 cells in a concentration- and time-dependent manner. The IC₅₀ values of sirtinol were 48.6 μM and 43.5 μM after 24 and 48 h of treatment, respectively (Fig. 1B).

The expression levels of SIRT1, SIRT2 and SIRT3 were measured by western blot analysis. The experiment revealed that sirtinol significantly decreased SIRT1 expression (Fig. 2A). Mammalian SIRT1 is the direct homologue of yeast SIRT2 and belongs to the NAD⁺-dependent HDAC family and has been implicated as a regulator of a variety of important biological processes, such as aging, metabolism and stress resistance (32). To further investigate the distinct effects of the SIRT inhibitor on cell fate, we measured the acetylated status of the SIRT1/2 target, p53. Sirtinol significantly increased the acetylated p53 level, whereas p53 acetylation was also accompanied by an induction of p53 stability (Fig. 2B).

To confirm that sirtinol affects cell cycle regulation in MCF-7 cells, we measured the cell cycle distribution using flow cytometry analyses. Sirtinol significantly induced G1 phase arrest at 48 h (Fig. 3A). The percentage of the sub-G1 population, which is indicative of apoptosis, increased after treatment with sirtinol in MCF-7 cells. We also examined the expression levels of cell cycle-regulated proteins by western blot analysis. Sirtinol significantly decreased the expression of cyclin B1, cyclin D1, CDK2 and CDK6, indicating that these molecules are closely associated with the G1 cell cycle check-point. Furthermore, sirtinol significantly increased p21 and p27 expression in MCF-7 cells (Fig. 3B).

To elucidate the mechanism underlying the cytotoxic effect of sirtinol on MCF-7 cells, apoptotic cell death was measured by Annexin V-FITC assay, DAPI staining and western blot analysis. Sirtinol (10 and 50 μM) significantly increased the population of late-stage apoptotic cells (Fig. 4A). DAPI staining was used to confirm the effect of sirtinol on apoptosis in MCF-7 cells. Sirtinol increased the number of apoptotic nuclei (condensed or fragmented chromatin) compared with the control culture, which showed enhanced fluorescence staining with DAPI (Fig. 4B). To determine the apoptotic pathway involved, we measured the expression of apoptosis-related protein levels by western blot analysis. Sirtinol (50 μM) significantly increased the cleavage of PARP, release of cytochrome c and the expression of Bax, and decreased the expression of Bcl-2 in MCF-7 cells (Fig. 4C). The activities of caspase 7 and caspase 9 were slightly increased in MCF-7 cells treated with sirtinol; however, caspase 8 activity was not altered (Fig. 4D).

To evaluate autophagic cell death induced by sirtinol, western blot analysis, acridine orange and MDC staining were performed. The conversion of the soluble form of LC3-I to the autophagic vesicle-associated form, LC3-II, is considered a specific marker of autophagosome promotion. Sirtinol significantly increased the level of LC3-II and decreased the unconjugated LC3-I levels. Moreover, similar to the LC3-II expression pattern, the level of beclin-1, known as Atg6, was increased by sirtinol treatment (Fig. 5A).

The induction of autophagy was confirmed by acridine orange and MDC staining. The vital dyes, acridine orange and MDC, are commonly used to study autophagy. Acridine orange is a lysotropic dye that accumulates in acidic organelles in a pH-dependent manner. At a neutral pH, acridine orange is a hydrophobic green fluorescent molecule. However, within acidic vesicles, acridine orange becomes protonated and trapped within the organelle and forms aggregates that emit bright red fluorescence (33). MDC is another popular autofluorescent marker that preferentially accumulates in autophagic vacuoles. While acridine orange staining in lysosomes is primarily due to ion trapping, MDC accumulation in auotphagic vacuoles is due to a combination of ion trapping and specific interactions with vacuole membrane lipids (34,35). With acridine orange staining, control cells primarily exhibited green fluorescence with minimal red fluorescence, indicating a lack of AVOs; however, sirtinol-treated cells showed a fold-increase in red fluorescent AVOs at 48 h (Fig. 5B). Flow cytometric analysis after acridine orange staining also showed an increase in red fluorescence intensity with sirtinol treatment, indicating an enhancement of AVOs (Fig. 5C). Histogram profiles show the mean fluorescence intensity of the control and drug-treated cells (Fig. 5D). Similar results were observed with MDC staining (Fig. 5E). There was significant autophagic vesicle formation in MCF-7 cells exposed to sirtinol. The morphological characteristics demonstrated that sirtinol induced autophagy in MCF-7 cells.

To explore whether the inhibition of autophagy sensitizes MCF-7 cells to sirtinol-induced apoptotic cell death, we used 3-methyladenine (3-MA), a well-known inhibitor of autophagy (36). 3-MA blocks autopha gic cell death by inhibiting phosphoinositide kinase-3 (PI3K), an enzyme required for autophagy. 3-MA alone had no toxic effect on MCF-7 cells. Following pre-treatment with 3-MA (0.1 mM), sirtinol significantly reduced cell viability in a dose-dependent manner (Fig. 6A). Western blot analysis was performed to assess the expression levels of autophagy- or apoptosis-related protein levels after 3-MA treatment. As shown in Fig. 6B, LC3-II, beclin 1, Atg5 and Atg7 levels were decreased in MCF-7 cells pre-treated with 3-MA compared with those in cells treated with sirtinol alone (Fig. 6B). In addition, 3-MA enhanced sirtinol-induced apoptosis. As shown in Fig. 6C, sirtinol increased the Bax level and decreased the Bcl-2 level in 3-MA pre-treated MCF-7 cells. The expression levels of caspase 7 and caspase 9 were also altered by sirtinol in the 3-MA-pre-treated MCF-7 cells (Fig. 6C). To confirm that apoptosis was affected by the inhibition of autophagy, cells were subjected to FITC-Annexin V/PI double-staining, followed by flow cytometric analysis to quantify the apoptotic cell populations. Sirtinol significantly increased the number of apoptotic cells among MCF-7 cells pre-treated with 3-MA (Fig. 6D).